Neurons transmit information via Ca2+-trigged exocytosis of synaptic vesicles. The synaptic vesicle protein synaptotagmin I has been proposed to serve as a major Ca 2+sensor that regulates release. Its cytoplasmic domain contains two Ca 2+sensing modules, C2A and C2B. Recent studies indicate that perturbation of the Ca2+-sensing ability of either the C2A or C2B domain inhibits the evoked fusion of docked synaptic vesicles. Furthermore, changes in the expression levels of different synaptotagmin isoforms alter the time-to-opening, as well as the dilation kinetics, of fusion pores. Thus, synaptotagmins are likely to interact with, and regulate, the membrane fusion machinery in vivo. However, the mechanism by which synaptotagmin regulates exocytosis remains to be established. A number of studies point to three potential effectors for the action of Ca2+ synaptotagmin: t-SNAREs (SNAP-25 and syntaxin), membranes, and other copies of synaptotagmin. A goal of this proposal is to determine whether these interactions function as coupling steps in exocytosis. The strategies of aims 1 and 2 are to selectively alter the kinetics and affinities of synaptotagmin-t-SNARE and synaptotagmin-membrane interactions, respectively. To test the hypothesis that these interactions trigger exocytosis, loss-of-function mutant copies of synaptotagmin will be expressed on the large dense core vesicles of PC12 cells where their effects on secretion will be monitored using amperometry. In aim 3 we will reconstitute full-length synaptotagmin into proteoliposomes in order to determine whether the kinetics of Ca2+-triggered oligomerization, within the plane of the bilayer, are rapid enough to couple Ca 2+to fusion. Once active protein is reconstituted, we will test the hypothesis that Ca2+-synaptotagmin acts to accelerate SNARE catalyzed membrane fusion by co-reconstituting synaptotagmin with purified SNAREs. This will provide a defined in vitro system to analyze the activities of selective loss-of-function mutants. A better understanding of exocytosis will provide a framework to determine how fusion is modulated (both pre- and post-synaptically) and thus contributes to synaptic plasticity in both normal and pathophysiological states. Finally, defining this mechanism should ultimately provide targets for treatment of diseases in which synaptic transmission is impaired.